EP1405862A2 - Verfahren zur Identifizierung antigener Peptide - Google Patents

Verfahren zur Identifizierung antigener Peptide Download PDF

Info

Publication number
EP1405862A2
EP1405862A2 EP03021521A EP03021521A EP1405862A2 EP 1405862 A2 EP1405862 A2 EP 1405862A2 EP 03021521 A EP03021521 A EP 03021521A EP 03021521 A EP03021521 A EP 03021521A EP 1405862 A2 EP1405862 A2 EP 1405862A2
Authority
EP
European Patent Office
Prior art keywords
cells
peptides
antigenic peptides
mhc
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP03021521A
Other languages
English (en)
French (fr)
Other versions
EP1405862A3 (de
EP1405862B1 (de
Inventor
Harald Kropshofer
Anne Vogt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
F Hoffmann La Roche AG
Original Assignee
F Hoffmann La Roche AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by F Hoffmann La Roche AG filed Critical F Hoffmann La Roche AG
Priority to EP03021521A priority Critical patent/EP1405862B1/de
Priority to EP07011493A priority patent/EP1826217B1/de
Priority to EP06015737A priority patent/EP1714981A3/de
Publication of EP1405862A2 publication Critical patent/EP1405862A2/de
Publication of EP1405862A3 publication Critical patent/EP1405862A3/de
Application granted granted Critical
Publication of EP1405862B1 publication Critical patent/EP1405862B1/de
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to methods useful for isolating antigenic peptides from a limited quantity of cells or bodily fluid from a mammalian organism in an amount sufficient to determine their sequence and identity. Therefore this invention relates to methods for identifying novel disease-associated antigens, e.g. tumor antigens and antigens involved in autoimmune diseases, to be utilized for diagnostic or therapeutic purposes.
  • the methods of the present invention can also be utilized for controlling the quality of vaccines. More specifically, the methods of the invention can be used for determining the sequence of antigenic peptides presented via peptide receptors of dendritic cells which are the most important antigen presenting cells of the body and valuable tools for vaccination.
  • Pathological conditions such as infectious diseases, autoimmune disorders or cancer
  • diseases can be distinguished from healthy conditions by the expression of disease-specific molecules.
  • proteins which are newly expressed, mutated or aberrantly expressed can be utilized as markers for the respective malignancy.
  • a potent class of markers serving as both diagnostic and therapeutic tools are protein fragments or peptides bound to molecules of the major histocompatibility complex (MHC).
  • MHC molecules are termed human leukocyte antigens (HLA).
  • HLA-associated peptides are short, encompassing 9-25 amino acids (Kropshofer, H. & Vogt, A.B., Immunol Today 18 (1997) 77-82).
  • these peptides are derived from self-proteins in order to establish self-tolerance.
  • HLA-associated peptides are derived from foreign proteins of viral, fungal or bacterial origin in order to fight foreign invaders.
  • T lymphocytes Through activation of specialized immune cells, named T lymphocytes (short: T cells), HLA-peptide complexes are indispensable for mounting a cellular or humoral immune response.
  • Particular self-peptides denoted autoantigenic peptides, are erroneously recognized by autoaggressive T cells giving rise to autoimmune diseases.
  • the lack of T cell recognition of self-peptides derived from tumor-specific antigens contributes to immune evasion and progressive growth of tumors (Boon, T. et al., Ann Rev Immunol. 12 (1994) 337-265).
  • increasing our knowledge about disease-associated marker peptides would be of considerable importance in tumor immunology and autoimmunity.
  • MHC-peptide complexes can be distinguished (Germain, R., Cell 76 (1994) 287-299): (i) MHC class I-peptide complexes can be expressed by almost all nucleated cells in order to attract CD8+ cytotoxic T cells which lyse infected cells or tumor cells, (ii) MHC class II-peptide complexes are constitutively expressed only on so-called antigen presenting cells (APCs), such as B lymphocytes, macrophages or dendritic cells (DCs). In particular, DCs have the capacity to prime CD4+ T helper cells (Banchereau, J. & Steinman, R.M., Nature 392 (1998) 245-254).
  • APCs antigen presenting cells
  • DCs have the capacity to prime CD4+ T helper cells (Banchereau, J. & Steinman, R.M., Nature 392 (1998) 245-254).
  • DCs can be licensed to optimally activate cytotoxic CD8+ T cells: this is accomplished through prior interaction of their MHC class II-peptide complexes with CD4+ T helper cells (Ridge, T. et al., Nature 393 (1998) 474-478).
  • peptides presented by MHC class II molecules on DCs play a superior role in the pathogenesis of diseases involving T cell-driven immune responses.
  • DCs differentiated and activated in vitro can be used for vaccination of cancer patients after co-culture with tumor cell-derived antigens or by employing analogous techniques. Pilot dendritic cell vaccination studies have successfully induced specific anticancer responses including clinical responses (Timmermann, J.M. & Levy, R., Ann Rev Medicine 50 (1999) 507-529; Nestle, F.O., et al., Nature Medicine 7 (2001) 761-765).
  • DC-based cancer cell vaccines comprise DCs pulsed with normal or gene-modified cancer cells, cancer cell lysates, cancer cells fused to DCs or cancer cell-derived heat shock protein- (Hsp-) peptide complexes.
  • Hsps derived from tumor cells carry tumor-specific peptides which are efficiently transferred onto MHC molecules of DCs.
  • These DCs finally prime cytotoxic T cells with anti-tumor reactivity which leads to the eradication of tumors in mice (Srivastava, P.K., et al., PNAS 83 (1986) 3407-3411; Suto, R., et al:, Science 269 (1995) 1585-1588; Binder, R.J. et al, Nature Immunol. 1 (2000) 151-162).
  • Vaccines based on the identification of cancer antigens include DCs primed with naked DNA, recombinant adeno- or vaccinia viruses, natural or recombinant proteins purified from the respective tumor cells or synthetic analogs of tumor peptides.
  • the advantage of pulsing DCs with antigenic tumor peptides rather than with genetic or protein precursors is that peptides can be loaded directly onto MHC molecules of DCs without further processing.
  • RA rheumatoid arthritis
  • RA patients suffer from systemic destruction of their joint tissue, which is mediated by auto-aggressive T lymphocytes and auto-antibodies.
  • the presence of both auto-reactive T cells and antibodies rely on the presentation of MHC class II-restricted peptide antigen.
  • HLA-DR molecules particularly the genes DRB1*0401 and DRB1*0404 in people of European descent, revealed to be major risk factors and confer increased susceptibility to RA (Marrack P et al. Nat. Med., 2002, 7: 899-905).
  • the present invention provides methods for isolating and identifying femtomolar amounts of peptide antigens presented by 0.1 to 5 ⁇ g MHC molecules or Hsp receptors isolated from an organism or from cells derived from an organism. Said methods concern immune monitoring of diseases, e.g. autoimmune diseases, the design of individualized peptide vaccines for the treatment of diseases, e.g. of cancer and the quality control of vaccines e.g. those based on dendritic cells.
  • the methods of the invention have the advantage that the identity of bound and/or presented antigenic peptides can be elucidated from very small amounts of bodily fluids or cells isolated from an mammalian organism.
  • the described methods ensure that the antigenic peptides isolated and identified are those that are bound and/or presented by peptide receptors in vivo or are those that are naturally-processed and presented by APCs, preferably DCs in vitro.
  • Fig. 1A is a diagram showing an overview of the methodology following strategy 1 (direct approach): MHC class II-peptide compelxes or Hsp-peptide complexes are isolated directly from tissue or bodily fluids thereby leading to the identification of naturally processed MHC class II or Hsp associated antigens presented in vivo.
  • Fig. 1B is a diagram showing an overview of the methodology following strategy 2 (indirect approach): Dendritic cells (DCs), the most specialized antigen presenting cells (APCs), are brought in contact with an antigenic source (e.g. bodily fluids) under optimal conditions for antigen uptake and antigen processing. As a control, DCs are cultured under the same conditions without contact with antigens. After maturation of DC antigen loaded MHC class II molecules are purified and the respective MHC class II associated antigenic peptides are isolated and identified.
  • DCs Dendritic cells
  • APCs antigen presenting cells
  • Fig. 2A illustrates strategy 2 and is a mass spectrometric analysis of HLA-DR bound peptides isolated from mature dendritic cells which were mock-treated (upper panel) or pulsed with the influenza vaccine Inflexal Berna VTM, containing virosome-encapsulated recombinant hemagglutinin from Influenza virus (lower panel).
  • the three major signals induced by treatment with Inflexal Berna VTM are marked by arrows and numbers.
  • Fig. 2B shows the protein sequence (one-letter-code) of the influenza hemagglutinin protein from strain B / Yamanashi / 166 / 98.
  • the newly identified HLA-DR epitope (cf. Fig. 2A) is underlined.
  • the present invention provides a method for isolating disease-associated antigenic peptides in femtomolar amounts allowing their identification which method comprises providing complexes of peptide receptors with antigenic peptides from a mammalian organism in an amount of 0.1 to 5 ⁇ g, preferably in an amount of 0.2 to 3 ⁇ g.
  • This quantity equals to the amount of material which is normally available from biopsies or bodily fluids of patients or healthy donors.
  • the lowest amount of material necessary in the prior art is about 200 ⁇ g MHC class II molecules derived from an unlimited source (inbred mice) (Dongre AR et al., EJI 2001, 31, 1485-94). This is about two orders of magnitude more material than available from human patient material.
  • the amount of tissue or bodily fluid necessary to obtain e.g. 100 ng MHC class II molecules depends on the number of cells that do express MHC class II and on the expression rate of MHC class II molecules: e.g. 100 ng of MHC class II are equivalent to about 2 x 10 5 mature DCs or 5 to 10 x 10 6 peripheral blood monocytes or about 5 x 10 7 peripheral blood mononuclear cells which can be obtained from about 50 ml of blood.
  • each type of these peptide receptors e.g. human MHC class II gene product HLA-DR1
  • HLA-DR1 carries about 500 to 1000 different antigenic peptides
  • most of the 500 to 1000 different peptides attain very low copy numbers and, therefore, are not very likely to play physiological role.
  • those peptides that are of immunological relevance e.g.
  • helper T cells those that activate helper T cells, attain moderate to high copy numbers (Latek RR & Unanue ER, Immunol. Rev. 1999, 172: 209-228).
  • These peptides cover about 40 to 50% of the total amount of peptide material eluted from MHC class II molecules and equal to about 10 to 200 individual peptides.
  • MHC class II associated peptides are represented as a set of 2 to 5 C- and N-terminal truncation variants (Rudensky AY et al, Nature 1992, 359, 429-431; Chicz et al. Nature 1992, 358: 764-768) sharing a common core sequence of about 10 to 13 amino acids which is essential for recognition by the T cell receptor.
  • These truncation /elongation variants constitute the same T cell epitope. This means that the number of different epitopes, which are of importance is actually smaller, ranging from about 5 to 70 different epitopes. Thus, the abundance of immunologically relevant epitopes ranges from 0.2% to 5%.
  • the method of the present invention comprises (a) providing complexes of peptide receptors with antigenic peptides isolated from a mammalian organism in an amount of 0.1 to 5 ⁇ g, and (b) eluting the associated antigenic peptides from the peptide receptors.
  • the antigenic peptides of the present invention are peptides which are associated with peptide receptors from tissue or body fluids or cells of a mammalian organism or from antigen presenting cells derived from a mammalian organism. They may be bound to transmembrane peptide receptors comprising MHC I and MHC II molecules presenting the antigenic peptides at the cell surface to T cells of the immune system. The antigenic peptides may also be bound to intra- or extracellular MHC molecules. Peptides may also be bound to intracellular peptide receptors relating to the heat shock protein (Hsp) family.
  • Hsp heat shock protein
  • the antigenic peptides comprise self antigens, tumor antigens, autoantigens, viral, bacterial, and parasitic antigens. Some antigenic peptides may induce tolerance. Other antigenic peptides may elicit an immune response and are therefore immunogenic peptides.
  • the antigenic peptides of the present invention may be naturally processed antigenic peptides that means they are generated from antigenic proteins by the proteolytic system of the respective cell and loaded onto peptide receptors.
  • the antigenic peptides may also be non-naturally processed synthetic or recombinant antigenic peptides which may have been administered to an organism where they have been loaded onto peptide receptors without further processing or they may have been contacted with cells expressing peptide receptors in cell culture or with isolated peptide receptors in vitro.
  • the methods of the present invention comprise antigenic peptides, which are naturally-processed antigenic peptides, as well as antigenic peptides which are synthetic or recombinant antigenic peptides.
  • each single peptide whose sequence has to be determined is represented in only femtomolar amounts.
  • 1 ⁇ g MHC class II (16 pmol) may carry dominant peptide species, with each single peptide attaining an occupancy of 0.1 - 2%, which equals to about 16 - 320 femtomoles.
  • the methods of the present invention allow the isolation of these femtomolar amounts of antigenic peptides from 0.1 to 5 ⁇ g of peptide receptors loaded with peptides and their subsequent sequencing.
  • the methods of the present invention relate to peptide receptors comprising all proteins binding antigenic peptides, comprising MHC class II molecules, MHC class I molecules and Hsp proteins.
  • complexes of peptide receptors with antigenic peptides may be isolated from diverse body fluids of an organism, e.g. of blood, serum, ascites, synovial fluid, from tissue samples of an organism, e.g. tumor biopsies, or from cells isolated from an organism.
  • Complexes of peptide receptors with antigenic peptides may be isolated from a mammalian organism, preferably from a human organism.
  • the peptide receptors are isolated from an organism in an amount of 0.1 to 5 ⁇ g. Preferably, the peptide receptors are isolated from an organism in an amount of 0.2 to 3 ⁇ g.
  • the methods of the present invention encompass all cells expressing peptide receptors, e.g. all cells comprising Hsp molecules with associated antigens, all cells comprising MHC molecules with associated antigens; cells expressing MHC I or Hsp molecules comprise nearly all nucleated cells; cells expressing MHC II molecules comprise B cells, macrophages, dendritic cells, monocytes, thymic epithelial cells, microglial cells, activated T cells and endothelial and epithelial cells after induction with pro-inflammatory cytokines e.g. IFNgamma.
  • the cells expressing MHC II molecules are also referred to as antigen presenting cells (APCs) (Unanue, E.R.. Macrophages, antigen presenting cells and the phenomena of antigen handling and presentation. In: Fundamental Immunology, 2nd edition (editor Paul, W.E) New York, Raven Press, 1989).
  • APCs antigen presenting cells
  • the membranes of the cells or tissue have to be solubilized.
  • Cell lysis may be carried out with methods known in the art, e.g. freeze-and-thaw cycles and the use of detergents, and combinations thereof.
  • Preferred lysis methods are solubilization using detergents, preferably TX-100, NP40, n-octylglucoside, Zwittergent, Lubrol, CHAPS, most preferably TX-100 or Zwittergent 3-12.
  • detergents preferably TX-100, NP40, n-octylglucoside, Zwittergent, Lubrol, CHAPS, most preferably TX-100 or Zwittergent 3-12.
  • Cell debris and nuclei have to be removed from cell lysates containing the solubilized receptor-peptide complexes by centrifugation. Therefore, in a further embodiment of the present invention, the complexes of peptide receptors with antigenic peptides are isolated from the cells with methods comprising solubilization with
  • the invention provides the purification of the MHC-peptide complexes from cell lysates by methods comprising immunoprecipitation or immunoaffinity chromatography.
  • immunoprecipitation or immunoaffinity chromatography antibodies specific for MHC class I or MHC class II molecules and suitable for these methods are used.
  • the specific antibodies are preferably monoclonal antibodies, and are covalently or non-covalently e.g. via Protein A, coupled to beads, e.g. sepharose or agarose beads.
  • a selection of the broad panel of anti-HLA antibodies used in the prior art comprises:
  • Monoclonal antibodies specific for different MHC class I and MHC class II molecules may be commercially obtained (e.g. Pharmingen, Dianova) or purified from the supernatant of the respective hybridoma cells using Protein A- or Protein G- affinity chromatography. Purified monoclonal antibodies may be coupled by various methods known in the art, preferably by covalently coupling antibody amino groups to CNBr-activated sepharose.
  • Immunoisolation of MHC molecules may be performed by incubating the antibody-beads with the cell lysate under rotation for several hours or chromatographically by pumping the cell lysate through a micro-column. Washing of the antibody-beads may be performed in eppendorf tubes or in the microcolumn. The efficacy of the immunoprecipitation may be analysed by SDS-PAGE and western blotting using antibodies recognizing denatured MHC molecules (anti-HLA-DRalpha: 1B5; anti-HLA class I: HC10 or HCA2).
  • Hsp-peptide complexes may be purified by methods known in the art (Binder, R. et al. J. Immunol., (2000), 165: 2582-2587). In brief, cells may be homogenized in a hypotonic buffer and fractionated by ammonium sulfate precipitation. The 50% precipitates may be applied to ADP-affinity beads and, subsequently, to DEAE anion exchange beads in ordered to purify Hsp70-peptide complexes. The 80% precipitates of the above ammonium sulfate precipitation may be used to purify Hsp90 family protein-peptide complexes by a combination of Concanavalin A affinity chromatography and DEAE anion exchange chromatography.
  • peptides By eluting the peptides from the receptor molecules, a complex mixture of naturally processed peptides derived from the source of potential antigen and from polypeptides of intra- or extracellular origin, is obtained. Only after elution, peptides can be fractionated and subjected to sequence analysis.
  • the antigenic peptides in the methods of the present invention may be eluted by a variety of methods known in the art, preferably by using diluted acid, e.g., diluted acetonitrile (Jardetzky TS et al., Nature 1991 353, 326-329), diluted acetic acid and heating (Rudensky AY et al., Nature 1991, 353, 622-626; Chicz RM et al., Nature 1992, 358, 764-768) or diluted trifluoro acetic acid at 37°C (Kropshofer H et al., J Exp Med 1992, 175, 1799-1803). Most preferably, the peptides are eluted at 37°C with diluted trifluoro acetic acid.
  • diluted acid e.g., diluted acetonitrile (Jardetzky TS et al., Nature 1991 353, 326-329), diluted acetic acid and heating (Rudensky AY et al.
  • the sequestered peptide receptor-peptide complexes are washed with water or low salt buffer before elution in order to remove residual detergent contaminants.
  • the low salt buffer may be a Tris, phosphate or acetate buffer in a concentration range of 0.5 - 10 mM, preferably in a concentration of 0.5 mM.
  • the peptide receptor-peptide complexes are washed with ultrapure water (sequencing grade) conventionally used for HPLC analysis, preferably with ultrapure (sequencing grade) water from MERCK. The washing step may be carried out by ultrafiltration.
  • the ultrafiltration may be carried out in an ultrafiltration tube with a cut-off of 30 kD, 20 kD, 10 kD or 5 kD, preferably of 30 kD and a tube volume of 0.5 - 1.0 ml ("Ultrafree" tubes; Millipore).
  • the washing in the ultrafiltration tube may be carried out 4 to 12 times, preferably 6 to 10 times, with a volume of 10 to 20 times the volume of the beads carrying the receptor-peptide complexes, preferably with a volume of 15 times the beads.
  • the eluted peptides may be separated from the remaining peptide receptor molecules using the same ultrafiltration tube. The eluted peptides may then be lyophilized.
  • the isolated antigenic peptides are fractionated, sequenced and identified.
  • sequencing it is understood that the amino acid sequence of the individual peptides in the mixture of isolated antigenic peptides is elucidated by methods adequate to sequence femtomolar amounts of peptides.
  • identifying it is understood that it is established from which proteins or polypeptides the antigenic peptides are derived and which sequence they constitute within these proteins or polypeptides.
  • the complex mixture of eluted peptides may be fractionated by one of a variety of possible chromatographic methods, e.g. by reversed phase, anion exchange, cation exchange chromatography or a combination thereof.
  • the separation is performed by C18-reverse phase chromatography or by reversed-phase / cation exchange two-dimensional HPLC, denoted as MudPit (Washburn MP et al., Nat Biotechnol., (2001), 19, 242-247).
  • the fractionation is done in a HPLC mode utilizing fused-silica micro-capillary columns which are either connected to a nano-flow electrospray source of a mass spectrometer or to a micro-fractionation device which spots the fractions onto a plate for MALDI analysis.
  • mass spectrometric techniques are suitable, preferably MALDI-post source decay (PSD) MS or electrospray ionization tandem mass spectrometry (ESI-MS), most preferably ion-trap ESI-MS.
  • PSD MALDI-post source decay
  • ESI-MS electrospray ionization tandem mass spectrometry
  • sequences of the individual peptides can be determined by means known in the art.
  • sequence analysis is performed by fragmentation of the peptides and computer-assisted interpretation of the fragment spectra using algorithms, e.g. MASCOT or SEQUEST. Both computer algorithms use protein and nucleotide sequence databases to perform cross-correlation analyses of experimental and theoretically generated tandem mass spectra. This allows automated high through-put sequence analysis.
  • MALDI-TOF matrix-assisted laser desorption and ionization time-of-flight
  • the run through of the micro-capillary column may be analyzed by a flow-through UV detector operated at a detection wave-length of 214 nm.
  • a flow-through UV detector operated at a detection wave-length of 214 nm.
  • the peak areas of peptides to be analyzed are compared with peak areas of graded amounts of synthetic standard peptides.
  • Strategy 1 of the present invention is used to isolate antigenic peptides which were loaded onto peptide receptors inside an organism (ex vivo approach, Fig. 1A).
  • the present invention relates to a method for isolating and identifying MHC or Hsp associated peptides in femtomolar amounts which method comprises providing 0.1 to 5 ⁇ g MHC-peptide or Hsp-peptide complexes from a mammalian organism.
  • This amount of peptide receptors equals to the amount of material which is normally available from biopsies or bodily fluids of patients or healthy donors, e.g. 100 ng of MHC class II-peptide complexes can be purified from about 5 x 10 7 peripheral blood mononuclear cells isolated from about 50 ml of blood.
  • the MHC-peptide or Hsp-peptide complexes may be purified from isolated cells e.g. blood monocytes, from a mixture of cells e.g. peripheral blood mononuclear cells, from tissue, e.g. tumor biopsies, or from body fluids e.g. ascites or synovial fluid.
  • isolated cells e.g. blood monocytes, from a mixture of cells e.g. peripheral blood mononuclear cells, from tissue, e.g. tumor biopsies, or from body fluids e.g. ascites or synovial fluid.
  • the body fluids may contain MHC-peptide or Hsp-peptide complexes bound to cells present in the body fluid, e.g. in synovial fluid, bound to vesicles present in the body fluid e.g. apoptotic vesicles or exosomes derived from cells (Denzer K et al., J Cell Science, 2000, 113, 3365-3374), or MHC-peptide complexes may be present in soluble form, due to shedding from the plasma membrane, e.g. soluble MHC class I and MHC class II molecules (Aultman D et al., Human Immunol., 1999, 60, 239-244).
  • MHC-peptide or Hsp-peptide complexes may be isolated from diverse body fluids of an organism, e.g. of blood, serum, ascites, synovial fluid or from tissue samples of an organism, e.g. biopsies, excised primary or secondary tumors or from cells isolated from an organisms.
  • Complexes of peptide receptors with antigenic peptides may be isolated from cells, tissue or body fluids from a mammalian organism, preferably from a human organism.
  • Strategy 2 of the present invention foresees isolation of antigenic peptides which have been loaded onto peptide receptors outside an organism, e.g. in cell culture (in vitro approach, Fig. 1B).
  • the present invention relates to a method for isolating antigenic peptides in femtomolar amounts which method comprises (a) providing MHC expressing cells in a numer providing 0.1 to 5 ⁇ g MHC molecules, (b) contacting the cells with a source of potential antigen, (c) isolating MHC molecule-antigenic peptide complexes from the cells and (d) eluting the associated peptides from the MHC molecules.
  • the MHC expressing cells may be MHC I or MHC II expressing cells (APCs).
  • APCs are dendritic cells, more preferably, the APCs are immature dendritic cells, most preferably, the APCs are immature dendritic cells generated from peripheral blood monocytes.
  • Dendritic cells may be generated from peripheral blood monocytes or from bone marrow-derived CD34+ stem cell-precursors.
  • the peripheral blood mononuclear cells (PBMCs) may be isolated from blood samples by density gradient centrifugation.
  • the monocytes may then be isolated from PBMCs by methods known in the art, e.g. by sorting with magnetic beads.
  • the source of dendritic cells may be mammalian species, preferably humans.
  • the monocytes may then be differentiated in cell culture to become immature dendritic cells. The differentiation state may be monitored by flow-cytometric analysis, e.g. using upregulation cell surface markers CD83, CD80, CD86, HLA-DR.
  • the amount of tissue or bodily fluid necessary to obtain e.g. 100 ng MHC class II molecules depends on the number of cells that do express MHC class II and on the expression rate of MHC class II molecules: e.g. 100 ng of MHC class II are equivalent to about 2 x 10 5 mature DCs or 5 to 10 x 10 6 peripheral blood monocytes or about 5 x 10 7 peripheral blood mononuclear cells which can be obtained from about 50 ml of blood.
  • the APCs are then contacted with a source of potential antigen.
  • the APCs preferably the immature dendritic cells, are at the same time triggered to mature by methods known in the art, e.g. incubation with inflammatory cytokines, like TNF alpha or a mixture of TNF alpha, IL-6, IL1beta PGE2.
  • the source of potential antigen offered to the APCs may be selected from the group comprising tumor tissue, tumor cells, tumor cell lines, gene-modified tumor cell lines, a crude cellular lysate of these cells or cell lines, tumor cell-derived heat shock proteins, pathogens, known viral, bacterial and parasitic antigens, tissues subject to immune attack, known self antigens, autoantigens, body fluids or tissue biopsies from patients with tumor, autoimmune or infectious diseases, body fluids or tissue biopsies from healthy individuals as reference controls. Control APCs are treated equivalently except that they are not exposed to a source of potential antigen.
  • the source of potential antigen may be derived from different mammalian species, preferably from human.
  • the APCs may be contacted with a source of potential antigen which is taken up by the APCs by receptor-mediated uptake or by fluid phase uptake and internalized.
  • the cells may also be infected with a source of potential antigen, e.g., with a virus.
  • the peptide sequences identified by the methods of the invention may be validated by one of several criteria, comprising MHC binding motif, MHC binding capacity and T cell recognition.
  • MHC binding motifs are common structural characteristics of peptides associated to a particular MHC molecule (allelic variant) which are necessary to form stable complexes with MHC molecules.
  • Peptide ligands eluted from MHC class I molecules are relatively short, ranging from 8 to 11 amino acids.
  • 2 or 3 side chains of the peptide are relevant for binding. The position of the respective amino acid side chains varies with the HLA allele, most often two of these so-called “anchor” residues are located at positions 2 and 9. With respect to a particular anchor position, only 1 or 2 amino acids normally can function as anchor amino acids e.g. leucine or valine V at position 2 in the case of HLA-A2.
  • the peptide length varies from 12 to 18 amino acids and even longer peptides can bind since both ends of the peptide binding groove are open.
  • Most HLA class II molecules accommodate up to 4 anchor residues at relative positions P1, P4, P6 and P9 contained in a nonameric core region. This core region, however, can have variable distance from the N-terminus of the peptide. In the majority of cases, 2-4 N-terminal residues precede the core region.
  • the P1 anchor residues is located at positions 3, 4 or 5 in most HLA class II associated peptides.
  • Peptides eluted from HLA-DR class II molecules share a big hydrophobic P1 anchor, represented by tyrosine, phenylalanine, tryptophane, methionine, leucine, isoleucine or valine.
  • the MHC binding capacity of the peptides identified by the methods of the present invention may be tested by methods known in the art using, for example, isolated MHC class II molecules and synthetic peptides with amino acid sequences identical to those identified by the method of the invention (Kropshofer H et al., J. Exp. Med. 1992; 175, 1799-1803; Vogt AB et al., J. Immunol. 1994; 153, 1665-1673; Sloan VS et al., Nature 1995; 375, 802-806).
  • a cellular binding assay using MHC class II expressing cell lines and biotinylated peptides can be used to verify the identified epitope (Arndt SO et al., EMBO J., 2000; 19, 1241-1251)
  • the relative binding capacity of a peptide is measured by determining the concentration necessary to reduce binding of a labelled reporter peptide by 50% (IC50). Peptide binding with a reasonable affinity to the relevant HLA class II molecules attains IC50 values not exceeding 10-fold the IC50 of established reference peptides.
  • binding assays can also be used to test the ability of peptides to bind to alternative class II MHC molecules, i.e., class II MHC molecules other than those from which they were eluted using the method of the invention.
  • T cell recognition may represent another epitope verification procedure involving testing of peptides identified by the methods of the invention for their ability to activate CD4+ or CD8+ T cell populations.
  • CD4+ T cell are activated by peptides binding to MHC class II molecules while CD8+ T cells are activated by peptides binding to MHC class I molecules.
  • Peptides with amino acid sequences either identical to those identified by the methods of the invention or corresponding to a core sequence derived from a nested group of peptides identified by the methods of the invention are synthesized.
  • the synthetic peptides are then tested for their ability to activate CD4+ (or CD8+) T cells from (a) test subjects expressing the MHC class II (or MHC class I) molecule of interest and having at least one symptom of the disease; and (b) control subjects expressing the MHC class II (or MHC class I) molecule of interest and having no symptoms of the disease. Additional control subjects can be those with symptoms of the disease and not expressing the MHC class II (or MHC class I) molecule of interest.
  • responsiveness in the CD4+ T cells of test subjects but not in CD4+ T cells of the control subjects described in (b) provides confirmatory evidence that the relevant peptide is an epitope that activates CD4+ T cells that can initiate, promote, or exacerbate the relevant disease.
  • diseases e.g., cancer or infectious diseases without an autoimmune component
  • a similar pattern of responsiveness and non-responsiveness to that described in the previous sentence would indicate that the relevant peptide is an epitope that activates CD4+ T cells that can mediate immunity to the disease or, at least, a decrease in the symptoms of the disease.
  • CD4+ (or CD8+) T cell responses can be measured by a variety of in vitro methods known in the art. For example, whole peripheral blood mononuclear cells (PBMC) can be cultured with and without a candidate synthetic peptide and their proliferative responses measured by, e.g., incorporation of [3H]-thymidine into their DNA. That the proliferating T cells are CD4+ (or CD8+) T cells can be tested by either eliminating CD4+ (or CD8+) T cells from the PBMC prior to assay or by adding inhibitory antibodies that bind to the CD4+ (or CD8+) molecule on the T cells, thereby inhibiting proliferation of the latter.
  • PBMC peripheral blood mononuclear cells
  • CD4+ (or CD8+) T cells are the proliferating cells.
  • CD4+ (or CD8+) T cells can be purified from PBMC and tested for proliferative responses to the peptides in the presence of APC expressing the appropriate MHC class II (or MHC class I) molecule.
  • APCs can be B-lymphocytes, monocytes, macrophages, or dendritic cells, or whole PBMC.
  • APCs can also be immortalized cell lines derived from B-lymphocytes, monocytes, macrophages, or dendritic cells.
  • the APCs can endogenously express the MHC class II (or MHC class I) molecule of interest or they can express transfected polynucleotides encoding such molecules. In all cases the APCs can, prior to the assay, be rendered non-proliferative by treatment with, e.g., ionizing radiation or mitomycin-C.
  • Cytokines include, without limitation, interleukin-2 (IL-2), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), TNF-alpha, interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12) or TGF-beta.
  • Assays to measure them include, without limitation, ELISA, and bio-assays in which cells responsive to the relevant cytokine are tested for responsiveness (e.g., proliferation) in the presence of a test sample.
  • cytokine production by CD4+ lymphocytes can be directly visualized by intracellular immunofluorescence staining and flow cytometry.
  • the methods of the present invention can be applied to identify peptides involved in the pathogenesis of a wide range of diseases, especially those in which susceptibility has been associated with expression of one or several particular MHC alleles or those where MHC-restricted T cell responses are lacking.
  • Candidate diseases include, without limitation, autoimmune diseases (e.g. rheumatoid arthritis (RA), type I diabetes, multiple sclerosis (MS), coeliac disease, myasthenia gravis (MG) and systemic lupus erythematosus (SLE)), cancer (e.g. melanoma, breast cancer, B cell lymphomas, prostate cancer, renal cancer) or infectious diseases (e.g. diseases caused by HIV, hepatitis C virus, measles virus, mycobacteria).
  • autoimmune diseases e.g. rheumatoid arthritis (RA), type I diabetes, multiple sclerosis (MS), coeliac disease, myasthenia gravis (MG) and systemic lupus erythematosus (SLE)
  • cancer e.g. melanoma, breast cancer, B cell lymphomas, prostate cancer, renal cancer
  • infectious diseases e.g. diseases caused by HIV, hepatitis C virus, measles
  • One aspect of the invention is a therapeutic purpose, wherein one or more of the identified peptides are used to vaccinate patients against cancer or infectious diseases.
  • the relevant peptides may be directly administered to the patient, in an amount sufficient for the peptides to bind to the MHC molecules, and provoke activation of T cells followed by T cell-mediated lysis of infected or cancer cells.
  • the relevant peptides may be utilized for the generation of vaccines based on DCs.
  • autologous DCs derived from patients' monocytes may be pulsed with the relevant peptides or recombinant proteins containing the relevant peptide sequences.
  • a combination of MHC class I- and class II-associated tumor antigenic peptides may be used to pulse DCs.
  • nucleic acid molecules which encode the relevant peptides may be incorporated into a vector in order to transfect tumor cells. These transfected tumor cells may be fused with DCs.
  • DCs presenting the relevant peptides in context of the appropriate MHC molecules will be administered to a patient for triggering a T cell response.
  • a further therapeutic application of the peptides relates to the situation where identified peptides are autoantigens in the context of autoimmune diseases.
  • complexes of the respective autoantigenic peptide and its restricting MHC class II molecules may be targeted by chimeric or humanized antibodies. This approach may lead to diminishment of autoantigenic MHC class II peptide complexes and, thus, to a decline in the number of autoaggressive CD4+ T helper cells which belong to one of the driving forces in autoimmunity.
  • the present invention provides the use of the described methods for the design of individualized peptide vaccines for the treatment of diseases, preferably cancer.
  • peptides of the invention may be used as response markers to track the efficacy of a therapeutic regime.
  • a therapeutic regime e.g. an autoantigenic peptide in the synovial fluid of rheumatoid arthritis patients
  • strategy 1 of the invention administer a given therapeutic drug and then monitor levels of the autoantigenic peptide thereafter, observing changes in peptide levels as indicia of the efficacy of the regime.
  • the present invention provides the use of the described methods for the control of the efficacy of a therapeutic treatment.
  • MHC-associated peptides derived from blood of patients suffering from autoimmune diseases may be used as response markers for therapeutic drugs against autoimmune diseases.
  • autoimmune diseases e.g. RA, type I diabetes, MS, coeliac disease, MG or SLE
  • response markers for therapeutic drugs against autoimmune diseases e.g. RA, type I diabetes, MS, coeliac disease, MG or SLE
  • autoantigenic peptides which are only found in certain stages or phases of an autoimmune disease may be utilized as stage-specific markers.
  • the present invention provides the use of the described methods for immune monitoring of diseases, preferably of autoimmune diseases.
  • a further aspect of the invention is a method for controlling the quality of vaccines based on DCs.
  • Autologous DCs used as vaccines against tumors are subjected to tumor antigens in order to load MHC molecules of DCs with appropriate tumor antigenic peptides.
  • a prerequisite of a high-quality DC vaccine is a high copy number of tumor antigenic peptides bound to the relevant MHC molecules.
  • the presence or absence of the relevant peptides can be tested prior to utilizing the respective DC preparations for vaccination.
  • the quality of vaccines based on other APCs e.g. macrophages or B cells, could be determined.
  • the present invention provides the use of the described methods for the quality control of vaccines.
  • the method of the invention not only allows the identification of MHC- or Hsp-associated peptides, but, at the same time, the protein wherefrom the peptide is derived. These proteins may be used as diagnostic markers in the very same manner as described above for the corresponding peptides.
  • the present invention provides a method of producing a pharmaceutical composition comprising the steps of the methods of the present invention, producing the identified peptides and optionally modifying them and formulating the product obtained with a pharmaceutically acceptable carrier or diluent.
  • compositions will contain an effective amount of the identified substance.
  • the antigenic peptide While it is possible for the antigenic peptide to be administered in a pure or substantially pure form, it is preferable to present it as a pharmaceutical composition, formulation or preparation.
  • compositions of the present invention both for veterinary and for human use, comprise an antigenic peptide as described above, together with one or more pharmaceutically acceptable carriers and, optionally, other therapeutic ingredients.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • the pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any method well-known in the pharmaceutical art.
  • All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation.
  • Formulations suitable for intravenous, intramuscular, subcutaneous, or intraperitoneal administration conveniently comprise sterile aqueous solutions of the active ingredient with solutions which are preferably isotonic with the blood of the recipient.
  • Such formulations may be conveniently prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride (e.g. 0.1-2.0M), glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile.
  • physiologically compatible substances such as sodium chloride (e.g. 0.1-2.0M), glycine, and the like
  • physiologically compatible substances such as sodium chloride (e.g. 0.1-2.0M), glycine, and the like
  • pH compatible substances such as sodium chloride (e.g. 0.1-2.0M), glycine, and the like
  • These may be present in unit or multi-dose containers, for example, sealed ampoules or vials.
  • the formulations of the present invention may incorporate a stabilizer.
  • Illustrative stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids which may be used either on their own or as admixtures. These stabilizers are preferably incorporated in an amount of about 0.11 to about 10,000 parts by weight per part by weight of immunogen. If two or more stabilizers are to be used, their total amount is preferably within the range specified above.
  • These stabilizers are used in aqueous solutions at the appropriate concentration and pH.
  • the specific osmotic pressure of such aqueous solutions is generally in the range of about 0.1 to about 3.0 osmoles, preferably in the range of about 0.8 to about 1.2.
  • the pH of the aqueous solution is adjusted to be within the range of about 5.0 to about 9.0, preferably within the range of 6-8.
  • anti-adsorption agent may be used.
  • RPMI 1640 medium (short: RPMI) supplemented with 1 mM Pyruvat, 2 mM Glutamine and 10% heat-inactivated fetal calf serum (Gibco BRL, Rockville, MD).
  • PBMCs peripheral blood mononuclear cells
  • Peripheral blood was obtained from the local blood bank as standard buffy coat preparations from healthy donors. Heparin (200 I.U./ml blood, Liquemine, Roche) was used to prevent clotting.
  • Peripheral blood mononuclear cells PBMCs were isolated by centrifugation in LSM® (1.077-1.080g/ml; ICN, Aurora, OH) at 800g (room temperature) for 30 min. PBMCs were collected from the interphase and washed twice in RPMI containing 20 mM Hepes (500g for 15 min, 300g for 5 min).
  • PBMCs were treated with ALT buffer (140 mM ammonium chloride, 20 mM Tris, pH 7.2) for 3 min at 37°C. PBMCs were washed twice with RPMI containing 20 mM Hepes (200g for 5 min).
  • ALT buffer 140 mM ammonium chloride, 20 mM Tris, pH 7.2
  • Monocytes were isolated from PBMCs by positive sorting using anti-CD14 magnetic beads (Miltenyi Biotech, Auburn, CA) according to the manufacturer's protocol. Monocytes were cultured in RPMI supplemented with 1% non-essential amino acids (Gibco, BRL, Rockville, MD), 50 ng/ml recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF; S.A. 1.1x10 7 U/mg) (Leucomax; Novartis, Basel Switzerland) and3 ng/ml recombinant human IL-4 (S.A. 2.9x10 4 U/mg) (R&D Systems, Minneapolis, MN). Monocytes were seeded at 0.3 x 10 6 /ml in 6-well plates (Costar) for 5 days to obtain immature dendritic cells.
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • CD1a high
  • CD3 low
  • CD19 low
  • CD56 low
  • CD80 low
  • CD83 high
  • CD86 low
  • HLA-DR high
  • mature dendritic cells display the following phenotype: CD1a (low), CD80 (high), CD83 (high), CD86 (high) and HLA-DR (high).
  • Monoclonal antibodies against CD1a, CD3, CD14, CD19, CD56, CD80, CD83, CD86 as well as the respective isotype controls were purchased from Pharmingen (San Diego, CA).
  • the anti-HLA-DR monoclonal antibody (mAb) L243 was produced by culturing the respective mouse hybridoma cell line.
  • mAb L243 was purified using ProteinA sepharose (Pharmacia, Uppsala, Sweden) and immobilized to CNBr-activated sepharose beads (Pharmacia) at a final concentration of 2.5 mg/ml, according to the manufacturer's protocol.
  • L243 beads were stored in PBS containing 0.1% Zwittergent 3-12 (Calbiochem, La Jolla, CA).
  • Pellets of frozen dendritic cells were resuspended in 10-fold volume of ice cold lysis buffer (1% Triton-X-100, 20 mM Tris, pH 7.8, 5 mM MgCl 2 , containing protease inhibitors chymostatin, pepstatin, PMSF and leupeptin (Roche, Mannheim, Germany)) and lysed in a horizontal shaker at 1000 rpm, 4°C for 1h. The cell lysate was cleared from cell debris and nuclei by centrifugation at 2000g, 4°C for 10 min.
  • ice cold lysis buffer 1% Triton-X-100, 20 mM Tris, pH 7.8, 5 mM MgCl 2 , containing protease inhibitors chymostatin, pepstatin, PMSF and leupeptin (Roche, Mannheim, Germany)
  • the lysate was co-incubated with L243 beads (5-10 ⁇ l L243 beads per 100 ⁇ l cell lysate) in a horizontal shaker at 1000 rpm, 4°C for 2 hrs.
  • Immunoprecipitated HLA-DR-peptide complexes bound to L243 beads were sedimented by centrifugation at 2000g, 4°C for 5 min and washed three times with 300 ⁇ l 0.1% Zwittergent 3-12 (Calbiochem ) in PBS.
  • HLA-DR-peptide complexes The efficacy of depletion of HLA-DR-peptide complexes was monitored by analyzing the respective cell lysates before and after immunoprecipitation. In parallel, aliquots of the beads were analyzed by western blotting using the anti-HLA-DRa-specific mAb 1B5 (Adams, T.E. et al., Immunology 50 (1983) 613-624).
  • HLA-DR-peptide complexes bound to L243 beads were resuspended in 400 ⁇ l H 2 O (HPLC-grade; Merck, Darmstadt, Germany), transferred to an ultrafiltration tube, Ultrafree MC, 30 kD cut-off (Millipore, Bedford, MA) and washed 10 times with 400 ⁇ l H 2 O (HPLC-grade) by centrifugation for 2-4 min at 14000 rpm at 4°C.
  • 50 ⁇ l 0.1% trifluoracetic acid Fluka, Buchs, Switzerland
  • H 2 O HPLC-grade
  • Lyophilized peptides eluted from HLA-DR molecules were resolved in 0.05% trifluoroacetic acid, 5% acetonitrile (Merck, Darmstadt, Germany) in H 2 O, (HPLC-grade) and separated on a 75 ⁇ m x 15 cm C18 PepMap capillary (C18; 3 ⁇ m; 100 ⁇ ) (LC-Packings, Amsterdam, Netherlands) connected to a FAMOSTM autosampler and an ULTIMATETM nano-flow HPLC (Dionex, Olten, Switzerland). The following non-linear gradient at a constant flow rate of 200 nl/min was used: 0-40min 5-50% system B; 40-50 min 50-90% system B.
  • System A was 0.05% trifluoroacetic, 5% acetonitrile/H 2 O and system B was 0.04% trifluoroacetic, 80% acetonitrile/H 2 O.
  • the separation was monitored via dual UV absorption at 214 nm and 280 nm.
  • Fractions (400 nl) were collected using the fraction collector PROBOTTM (BAI, Rothstadt, Germany) and spotted onto an AnchorChip 600/384 MALDI-MS target (Bruker, Bremen, Germany).
  • MudPIT multidimensional protein identification technology
  • the lyophilized peptides eluted from HLA molecules were resuspended in a buffer containing 5% (v/v) acetonitrile, 0.5% (v/v) acetic acid, 0.012% (v/v) heptafluoro butyric acid (HFBA) and 5% (v/v) formic acid.
  • the sample was separated on a fused-silica microcapillary column (100 ⁇ m i.d. ⁇ 365 ⁇ m) generated by a Model P-2000 laser puller (Sutter Instrument Co., Novato, CA).
  • the microcolumn was packed with 3 ⁇ m / C18 reverse-phase material (C18-ACE 3 ⁇ m [ProntoSIL 120-3-C18 ACE-EPS, Leonberg, Germany]) followed by 3 cm of 5 ⁇ m cation exchange material (Partisphere SCX;Whatman, Clifton, NJ).
  • a fully automated 8-step gradient separation on an Agilent 1100 series HPLC was carried out, using the following buffers: 5% ACN/0.02% HFBA/0.5% acetic acid (buffer A), 80% ACN/0.02% HFBA/0.5% acetic acid (buffer B), 250 mM ammonium acetate/5% ACN/0.02% HFBA/0.5% acetic acid (buffer C), and 1.5 M ammonium acetate/5% ACN/0.02% HFBA/0.5% acetic acid (buffer D).
  • the first step of 106 min consisted of a 100 min gradient from 0 to 80% buffer B and a 6 min hold at 80% buffer B.
  • the next 6 steps are characterized by the following profile: 5 min of 100% buffer A, 2 min of x% buffer C, 5 min of 100% buffer A, a 3 min gradient from 0 to 10% buffer B, a 55 min gradient from 10 to 35% buffer B, a 20 min gradient from 35 to 50% buffer B, a 16 min gradient from 50 to 80% buffer B.
  • the 2 min buffer C percentages (x) in steps 2-7 were as follows: 10, 20, 30, 40, 70, 90, and 100%.
  • Step 8 consisted of the following profile: a 5 min 100% buffer A wash, a 20 min salt wash with 100% buffer D and a 100 min gradient from 0-80% buffer B.
  • the HPLC column was directly coupled to a Finnigan LCQ ion trap mass spectrometer (Finnigan, Bremen, Germany) equipped with a nano-LC electrospray ionization source. Mass spectrometry in the MS-MS mode was performed according to the manufacturer's protocol. The identification of peptides was done by the sequest algorithm against the swiss.fasta database.
  • MALDI-PSD analysis was performed on a Bruker Ultraflex TOF/TOF mass spectrometer (Bruker, Bremen, Germany) using the software FLEXControl 1.1 Alpha for data acquisition. Calibration was achieved by using a tryptic digest of human serum albumin (Merck, Darmstadt, Germany). Peptide mixtures were first scanned in a reflectron mode. Peptides of interest were then selected for lift mode (MALDI-PSD analysis).
  • the peptide fragmentation spectra obtained were automatically evaluated using the Xmas 5.1.2 and Biotools 2.1 Software (Bruker) and used for sequence identification in a non-redundant protein database using the MASCOT algorithm (http://www.matrixscience.com).
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • T lymphocytes about 50%
  • B lymphocytes 5-10%)
  • monocytes 15-25%
  • natural killer cells about 6%
  • Peripheral blood dendritic cells are also present but only in very low amounts ( ⁇ 0.5%).
  • Analysis of PBMCs by flow cytometry revealed that both B cells and monocytes express considerable amounts of HLA-DR molecules, while natural killer cells and T cells stain negative. The small amount of dendritic cells in PBMCs cannot be visualized by FACS. Human T cells are able to up-regulate HLA-DR upon activation, however, activated T cells are normally absent from peripheral blood.
  • the number of B cells present in PBMCs is 2 to 3-fold lower compared to monocytes, their HLA-DR expression level is about 2-fold higher. This means that in lysates from PBMCs the number of HLA-DR molecules originating from B cells is comparable to the number of HLA-DR molecules from monocytes.
  • PBMCs were lysed in TX-100 and HLA-DR molecules were precipitated using anti-DR mAb L243. Precipitation was controlled by western blot analysis using anti-DRalpha mAb 1B5. Quantitative western blot analysis using purified HLA-DR molecules as a reference revealed that about 200 ng HLA-DR was purified from 5.3 x 10 7 cells. HLA-DR associated peptides were eluted in 0.1% TFA and the peptide mixture was fractionated using 2-dimensional cation-exchange/ reversed phase liquid chromatography (MudPit). Sequencing was done by high-throughput ion trap mass spectrometry and data base search was performed using human data bank "humangp". The peptides identified with a cross-correlation > 2.0 are listed in Table 1.
  • 27 peptides could be identified: 8 peptides were derived from human serum albumin and constituted a nested set of peptides typical for MHC class II associated peptides (N- and C-terminal elongation / truncation variants of the same epitope); 3 peptides were derived from apolipoprotein AII, again representing only one epitope; 3 peptides were derived from alphal anti-trypsin and represented one epitope; 4 peptides from protein disulfide isomerase related protein ERp72 (1 epitope). The last 9 peptides were derived from different proteins and, thus, represented 9 different epitopes.
  • peptides (4 epitopes) were derived from extracellular proteins that are major constituents of human serum: serum albumin is the most abundant protein in serum, apolipoprotein AII is a constituent of high density lipoproteins (HDLs), alpha 1 anti-trypsin is a well known serum protease inhibitor.
  • Ferritin light chain (donor of peptide nr. 10) is present in virtually all cells and at low concentrations in plasma. Most likely, these proteins were internalized by fluid phase uptake, and after proteolytic processing the respective peptides were loaded onto HLA-DR molecules inside the APC (monocyte or B cell). Alternatively, these proteins or fragments of the respective proteins could have bound to cell surface HLA-DR molecules.
  • the lysosomal-associated multi-transmembrane protein (lam5) is the donor of peptide nr. 4 and is expressed in haematopoietic cells.
  • the subcellular localization of lam 5 is the lysosome, so it is already present in the loading compartment of HLA-DR molecules, where it can bind before or after proteolytic cleavage.
  • HLA class I molecules (giving rise to peptide nr. 19) are present in nearly all nucleated cells, thus it is likely to be derived from the APC itself. Alternatively, HLA class I can be taken up from the serum where shedded HLA class I molecules have been described.
  • PDI ERp72 is an ER resident protein that has been described to be expressed in muscle and lung but also in a lymphoblastoid cell line. ERp72 gave rise to peptides nr. 20 to 23.
  • Pyruvate kinase is a cytosolic protein and exists in several isoforms with isoform M1 being expressed in muscle, heart and brain, and isoform M2 is described for fetal tissue.
  • the epitope (peptides 20-23) is present in both isoforms.
  • Peptide nr. 24 is derived from actin alpha 1, a cytosolic protein that is highly expressed in skeletal muscle. Thus, all three epitopes could be derived from muscle cells that might have released their cellular proteins into the serum due to micro-tissue injury or damage.
  • Peptide nr. 5 is derived from F-box helicase 1, however, little is known, as yet, about the tissue expression of this protein.
  • Peptides nr. 25, 26, 27 are derived from proteins that could be allocated to chromosomes 17, 6 and 4, respectively, but there was no information about the respective proteins or their function, tissue expression or subcellular localization.
  • the bovine analogue (TPTLVEVSRSLGKVGTR) of the serum albumin epitope (peptides nr. 11-18) has been described as a HLA-DR-associated epitope in context of the DR alleles DRB1*1101/ DRB1*1104 (Verreck FA et al., Immunogenetics 1996; 43, 392-397). It has been identified by Edman sequencing of self-peptides derived from cultured EBV-transformed B cells. The peptide binding motif of both of these alleles requires an aromatic or aliphatic residue at position P1, an aliphatic residue at P4 and a basic residue at P6 (Verreck FA et al., Immunogenetics 1996; 43, 392-397).
  • Fig. 1B was used to identify novel peptides associated to HLA-DR molecules expressed on the surface of dendritic cells that are exposed to a potential antigen.
  • the antigenic source was a commercially available vaccine against influenza virus, denoted as INFLEXAL Berna V (Berna, Bern, Switzerland).
  • Dendritic cells were differentiated from peripheral blood monocytes and cultured in a concentration of 0.5 x 10 6 cells/ml. 6 x 10 6 dendritic cells were exposed to the vaccine INFLEXAL Berna V (Berna, Bern, Switzerland) for 24 hrs by adding INFLEXAL Berna V at a concentration of 100 ⁇ l/ml (corresponding to 3 ⁇ g/ml hemagglutinin derived from influenza virus). At the same time maturation of dendritic cells was induced by adding TNFalpha (10 ng/ml). As a control, the same amount of dendritic cells (6 x 10 6 ) was cultured in the absence of the antigen, but in then presence of TNFalpha (10 ng/ml).
  • HLA-DR associated peptides were eluted with 0.1% TFA and analyzed by MALDI-MS (Fig. 2A): The upper panel shows the complex mixture of HLA-DR-associated self-peptides from unpulsed DCs.
  • the hemagglutinin proteins contained in INFLEXAL Berna V are derived from 3 different influenza strains: strain A / New Caledonia / 20 / 99; strain A / Panama / 2007 /99 ; strain B / Yamanashi / 166 / 98 (according to the recommendation of the WHO taking into account the genetic diversity among the circulating viruses (Lindstrom SE et al., J. Virol. 1999, 73, 4413-4426)).
  • KPGKTKTIVYQRGILLPQK contains the MHC peptide binding motif for DRB1*0101 and DRB5*0101 using I-260 as P1, Q-264 as P4 and L269 as P9 anchor residue (anchor residues are underlined).
  • strategy 2 (Fig. 1B) was used to identify novel HLA-DR-associated tumor peptides.
  • dendritic cells were exposed to a necrotic melanoma cell line, UKRV-Mel-15a.
  • 3 x 10 6 cells dendritic cells were co-incubated with 9 x 10 6 necrotic cells of the melanoma line UKRV-Mel-15a and cultured for 24 hrs in presence of TNFalpha (10 ng/ml).
  • TNFalpha 10 ng/ml
  • 3 x 10 6 cells dendritic cells were cultured in presence TNFalpha (10 ng/ml) only.
  • the HLA-DR associated peptides from both DC cultures were compared by MALDI-MS spectrometry and only the peptide signals contained in the profile of DCs pulsed with melanoma cells were used to identify new epitopes by successive sequencing.
  • Vimentin(202-217) and several other known melanoma antigens share a common motif suitable for binding to HLA-DR4 B1*0401 molecules (Table 2).
  • the peptides derived from melanA, CDC27, tyrosinase and vimentin display asparartic acid (D) instead of threonine (T) or serine (S) at anchor position P6. The relevance of this peculiarity remains to be investigated.
  • Vimentin is known to be a marker protein in a variety of benign and malign tumors. Together with melanA/MART-1, tyrosinase and S100, vimentin is routinely used to trace melanoma cells in clinical specimens from melanoma patients. Interestingly, melanoma clones with low invasive potential have a high vimentin expression, whereas vimentin is downregulated in highly invasive melanoma cell clones (Gutgemann A et al., Arch Dermatol Research 2001; 293, 283-290). In contrast, enhanced expression of vimentin is observed in poorly differentiated and metastatic prostate carcinoma (Lang SH et al., Prostate 2002; 52, 253-263).
  • vimentin is overexpressed in human renal cell carcinoma in relation to normal renal tissue (Stassar MJ et al. Br. J. Cancer 2001; 85, 1372-1382). Likewise, >95% of tumor cells in classical Hodgkin's lymphoma are vimentin positive, whereas T-cell-rich B-cell lymphomas are negative for vimentin (Rudiger T et al., Am J Surg Path 1998, 22, 1184-91).
  • Vimentin(202-217) peptide identified by the method of the invention is the first vimentin derived HLA class II restricted epitope described so far.
  • Fig. 1B was used to identify as many peptides as possible which were bound to HLA-DR molecules of dendritic cells (DCs) after TNFalpha-induced maturation and exposure to potential antigens. Sequencing was done by high-throughput ion trap MS/MS technology.
  • 5 x 10 6 cells dendritic cells were co-incubated with 1.5 x 10 7 necrotic cells of the melanoma line UKRV-Mel-20c and cultured for 24 hrs in presence of TNFalpha (10 ng/ml).
  • 5 x 10 6 cells dendritic cells were cultured in presence TNFalpha (10 ng/ml) only.
  • Both sets of dendritic cells were lysed in detergent TX-100 and HLA-DR molecules were precipitated using anti-DR mAb L243.
  • HLA-DR associated peptides were eluted with 0.1% TFA and analyzed by LC-high-throughput ion trap MS/MS technology.
  • 9 melanoma cell induced epitopes are derived from known tumor marker proteins, namely translation factor IF-4A1, translation factor EF-1alpha and interferon-gamma (IFNgamma)-inducible P78.
  • the translation initiation factor IF-4A1 is consistently overexpressed in melanoma cell lines in relation to normal human melanocytes. IF-4A1 overexpression seems to be an important feature of melanoma cells and might contribute to their malignant transformation (Eberle J et al., Int. J. Cancer 1997; 71, 396-401).
  • EF-1alpha ribosomal elongation factors
  • Prostate cancer progression from a hormon-dependent to a hormon-independent state includes a cascade of genetic alterations caused by activation of oncogenes and/ or inactivation of tumor suppressor genes.
  • Several genes were identified which are highly overexpressed in androgen-independent cancer cell lines.
  • the interferon-inducible genes 1-8U and p78 were identified (Markku H et al., Lab Invest. 2000; 80, 1259-1268.).
  • the peptides derived from translation factor IF-4A1, translation factor EF-1alpha and IFNgamma-inducible P78 identified by the method of the invention are new candidate tumor antigens that can be used as diagnostic markers or therapeutic vaccines.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Oncology (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Communicable Diseases (AREA)
  • Virology (AREA)
  • Diabetes (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Endocrinology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Emergency Medicine (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Transplantation (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • AIDS & HIV (AREA)
  • Reproductive Health (AREA)
EP03021521A 2002-10-02 2003-09-24 Verfahren zur Identifizierung antigener Peptide Expired - Lifetime EP1405862B1 (de)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP03021521A EP1405862B1 (de) 2002-10-02 2003-09-24 Verfahren zur Identifizierung antigener Peptide
EP07011493A EP1826217B1 (de) 2002-10-02 2003-09-24 Verfahren zur Identifizierung antigener Peptide
EP06015737A EP1714981A3 (de) 2002-10-02 2003-09-24 Verfahren zur Identifizierung antigener Peptide

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP02022223 2002-10-02
EP02022223 2002-10-02
EP03021521A EP1405862B1 (de) 2002-10-02 2003-09-24 Verfahren zur Identifizierung antigener Peptide

Related Child Applications (2)

Application Number Title Priority Date Filing Date
EP06015737A Division EP1714981A3 (de) 2002-10-02 2003-09-24 Verfahren zur Identifizierung antigener Peptide
EP07011493A Division EP1826217B1 (de) 2002-10-02 2003-09-24 Verfahren zur Identifizierung antigener Peptide

Publications (3)

Publication Number Publication Date
EP1405862A2 true EP1405862A2 (de) 2004-04-07
EP1405862A3 EP1405862A3 (de) 2004-06-09
EP1405862B1 EP1405862B1 (de) 2008-04-09

Family

ID=32116218

Family Applications (3)

Application Number Title Priority Date Filing Date
EP03021521A Expired - Lifetime EP1405862B1 (de) 2002-10-02 2003-09-24 Verfahren zur Identifizierung antigener Peptide
EP07011493A Expired - Lifetime EP1826217B1 (de) 2002-10-02 2003-09-24 Verfahren zur Identifizierung antigener Peptide
EP06015737A Withdrawn EP1714981A3 (de) 2002-10-02 2003-09-24 Verfahren zur Identifizierung antigener Peptide

Family Applications After (2)

Application Number Title Priority Date Filing Date
EP07011493A Expired - Lifetime EP1826217B1 (de) 2002-10-02 2003-09-24 Verfahren zur Identifizierung antigener Peptide
EP06015737A Withdrawn EP1714981A3 (de) 2002-10-02 2003-09-24 Verfahren zur Identifizierung antigener Peptide

Country Status (8)

Country Link
US (1) US20040086521A1 (de)
EP (3) EP1405862B1 (de)
JP (3) JP4620339B2 (de)
AT (2) ATE474851T1 (de)
AU (1) AU2003248438A1 (de)
CA (1) CA2441690C (de)
DE (2) DE60320196T2 (de)
ES (1) ES2346574T3 (de)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1715343A1 (de) 2005-04-20 2006-10-25 F. Hoffmann-La Roche Ag Verfahren zur Identifizierung von Epitopen in Zusammenhang mit Immunogenität bei Biopharmazeutika
CN105928771A (zh) * 2016-04-08 2016-09-07 四川自豪时代药业有限公司 一种检测疫苗抗原含量的样品前处理方法

Families Citing this family (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE442377T1 (de) * 2003-08-07 2009-09-15 Hoffmann La Roche Ra antigene peptide
ES2334936T3 (es) * 2005-04-20 2010-03-17 F. Hoffmann-La Roche Ag Metodo de identificacion de epitopos relacionados con la inmunogenicidad en biofarmacos.
JPWO2007055283A1 (ja) * 2005-11-10 2009-04-30 Jsr株式会社 核内受容体タンパク質複合体のプロテオミクス解析方法
US20100086938A1 (en) * 2006-11-29 2010-04-08 Takashi Shimada Mass spectrometry of biological sample using immunoprecipitation
ES2663662T3 (es) * 2008-01-18 2018-04-16 President And Fellows Of Harvard College Métodos para detectar distintivos de enfermedades o afecciones en fluidos corporales
JP5823379B2 (ja) * 2009-05-29 2015-11-25 ノバルティス アーゲー インフルエンザウイルスヘマグルチニンについてのアッセイ
WO2012012717A1 (en) 2010-07-23 2012-01-26 President And Fellows Of Harvard College Methods of detecting prenatal or pregnancy-related diseases or conditions
US20130184178A1 (en) 2010-07-23 2013-07-18 President And Fellows Of Harvard College Methods of Detecting Autoimmune or Immune-Related Diseases or Conditions
WO2013150680A1 (ja) * 2012-04-06 2013-10-10 独立行政法人産業技術総合研究所 プロテインタグ、タグ化タンパク質及びタンパク質精製方法
WO2014164366A1 (en) 2013-03-09 2014-10-09 Harry Stylli Methods of detecting cancer
EP3152574A4 (de) * 2014-06-06 2017-12-06 Singapore Health Services Pte Ltd Verfahren zur verwendung von zellen in zusammenhang mit einer autoimmunkrankheit
KR101831483B1 (ko) 2014-06-17 2018-02-23 이화여자대학교 산학협력단 Mhc-페리틴 결합체, 이를 포함하는 나노파티클 및 이의 용도
US10718781B2 (en) 2014-07-14 2020-07-21 Chugai Seiyaku Kabushiki Kaisha Method for identifying epitope on protein
WO2016079143A1 (de) 2014-11-17 2016-05-26 Pharis Biotec Gmbh Inhibitor der viralen hepatitis c infektion
CN110118845A (zh) * 2018-02-06 2019-08-13 北京金则医学科技发展有限公司 检测多种氨基酸和酰基肉碱、甲基丙二酸的方法和试剂盒
JP6712369B2 (ja) * 2018-05-29 2020-06-24 株式会社森永生科学研究所 抗ペプチド抗体の製造方法及び設計方法
US11603565B2 (en) * 2018-06-15 2023-03-14 Roche Sequencing Solutions, Inc. System for identification of antigens recognized by T cell receptors expressed on tumor infiltrating lymphocytes
WO2020037046A1 (en) * 2018-08-14 2020-02-20 Board Of Regents, The University Of Texas System Single molecule sequencing peptides bound to the major histocompatibility complex
CN113167796A (zh) * 2018-09-11 2021-07-23 朱诺治疗学股份有限公司 对工程化细胞组合物进行质谱分析的方法
JP2021047198A (ja) * 2020-12-04 2021-03-25 Ｋａｒｙｄｏ ＴｈｅｒａｐｅｕｔｉＸ株式会社 心筋梗塞、認知症、および腫瘍からなる群から選択される一種を予防、または治療するための有効成分の候補物質のスクリーニング装置、スクリーニングプログラム、およびスクリーニング方法
CN113406240B (zh) * 2021-07-02 2023-03-24 杭州艾儿默细胞生物科技有限公司 检测pHLA复合物中抗原肽的超滤-高效液相色谱法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002040647A1 (en) * 2000-11-14 2002-05-23 U.S. Army Medical Research Institute Of Infectious Diseases Method of establishing cultures of human dendritic cells and use thereof
US6455048B1 (en) * 1997-02-07 2002-09-24 Fordham University Prevention of primary and metastatic neoplastic diseases with hsp70-peptide complexes

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000005249A2 (en) * 1998-07-23 2000-02-03 The President And Fellows Of Harvard College Synthetic peptides and methods of use for autoimmune disease therapies
EP1098902A4 (de) * 1998-07-23 2002-07-24 Yeda Res & Dev Behandlung von autoimmunkrankheiten mittels copolymer 1 und ähnlichen copolymeren und peptiden

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6455048B1 (en) * 1997-02-07 2002-09-24 Fordham University Prevention of primary and metastatic neoplastic diseases with hsp70-peptide complexes
WO2002040647A1 (en) * 2000-11-14 2002-05-23 U.S. Army Medical Research Institute Of Infectious Diseases Method of establishing cultures of human dendritic cells and use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KROPSHOFER H ET AL.: "Self-Release of CLIP in Peptide Loading of HLA-DR Molecules" SCIENCE, vol. 270, no. 5240, 1995, pages 1357-1359, XP008026441 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1715343A1 (de) 2005-04-20 2006-10-25 F. Hoffmann-La Roche Ag Verfahren zur Identifizierung von Epitopen in Zusammenhang mit Immunogenität bei Biopharmazeutika
CN105928771A (zh) * 2016-04-08 2016-09-07 四川自豪时代药业有限公司 一种检测疫苗抗原含量的样品前处理方法

Also Published As

Publication number Publication date
JP2011012073A (ja) 2011-01-20
DE60333500D1 (de) 2010-09-02
EP1826217A1 (de) 2007-08-29
EP1714981A3 (de) 2006-12-20
EP1405862A3 (de) 2004-06-09
JP2004123749A (ja) 2004-04-22
EP1714981A2 (de) 2006-10-25
DE60320196T2 (de) 2009-05-14
CA2441690C (en) 2011-08-02
US20040086521A1 (en) 2004-05-06
AU2003248438A1 (en) 2004-04-22
CA2441690A1 (en) 2004-04-02
EP1826217B1 (de) 2010-07-21
DE60320196D1 (de) 2008-05-21
JP4620339B2 (ja) 2011-01-26
EP1405862B1 (de) 2008-04-09
ES2346574T3 (es) 2010-10-18
ATE391723T1 (de) 2008-04-15
ATE474851T1 (de) 2010-08-15
JP2007211020A (ja) 2007-08-23

Similar Documents

Publication Publication Date Title
CA2441690C (en) Method for the identification of antigenic peptides
US20070073042A1 (en) Novel MHC II associated peptides
JP5627180B2 (ja) 無差別的にヒト白血球抗体（ｈｌａ）クラスｉｉ分子に結合する腫瘍関連ペプチド
US8211436B2 (en) Anticancer vaccine and diagnostic methods and reagents
Halder et al. Isolation of novel HLA-DR restricted potential tumor-associated antigens from the melanoma cell line FM3
US20060251664A1 (en) Method for the identification of epitopes related to immunogenicity in biopharmaceuticals
EP1654272B1 (de) Ra antigene peptide
US6521598B1 (en) H-Y antigen
AU2006200569B2 (en) Method for the identification of antigenic peptides
EP1715343B1 (de) Verfahren zur Identifizierung von Epitopen in Zusammenhang mit Immunogenität bei Biopharmazeutika
JP5736333B2 (ja) 無差別的にヒト白血球抗体（ｈｌａ）クラスｉｉ分子に結合する腫瘍関連ペプチド
JP5736334B2 (ja) 無差別的にヒト白血球抗体（ｈｌａ）クラスｉｉ分子に結合する腫瘍関連ペプチド
Moore et al. Low abundance of naturally processed T-cell determinants following antigen pulsing of antigen-specific human B-cell lines.

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

PUAL Search report despatched

Free format text: ORIGINAL CODE: 0009013

AK Designated contracting states

Kind code of ref document: A3

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

17P Request for examination filed

Effective date: 20041209

AKX Designation fees paid

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

17Q First examination report despatched

Effective date: 20050803

RIC1 Information provided on ipc code assigned before grant

Ipc: A61K 39/00 20060101ALI20070627BHEP

Ipc: C07K 14/47 20060101AFI20070627BHEP

Ipc: A61K 38/17 20060101ALI20070627BHEP

Ipc: G01N 33/68 20060101ALI20070627BHEP

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REF Corresponds to:

Ref document number: 60320196

Country of ref document: DE

Date of ref document: 20080521

Kind code of ref document: P

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080409

NLV1 Nl: lapsed or annulled due to failure to fulfill the requirements of art. 29p and 29m of the patents act
PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080409

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080720

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080909

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080709

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080409

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080409

EN Fr: translation not filed
PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080409

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080709

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080409

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080409

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080409

Ref country code: BE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080409

26N No opposition filed

Effective date: 20090112

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20080930

Ref country code: FR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20090130

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080409

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20080924

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080409

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080409

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20081010

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20080924

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080409

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20080710

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20220810

Year of fee payment: 20

Ref country code: DE

Payment date: 20220615

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 20221001

Year of fee payment: 20

REG Reference to a national code

Ref country code: DE

Ref legal event code: R071

Ref document number: 60320196

Country of ref document: DE

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: GB

Ref legal event code: PE20

Expiry date: 20230923

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION

Effective date: 20230923